The Effect Of Sphingomyelin Synthase 2 On Sphingolipids Metabolisim And Physiologic Function In INS-1 Cell

Chapter I the effect of SMS2 expression level on the sphingolipids metabolism in INS-1 cellObjective:To construct the SMS2 overexpression and knockdown stable insulinoma cell lines and to detect its infection effectiveness in rat insulinoma cell(INS-1).It is the basis of functional study of SMS2in rat islet beta cell.Methods:The interference gene sequence of rat SMS2 was designed and was then used to build pL/shRNA/GFP-SMS2 plasmid.Meanwhile,human SGMS2 sequence was selectively amplified and inserted into pL/To/V5/GFP/IRES vector.The INS-1 cells were infected by the two concentrated lentivirus,respectively,which were expressed by 293T transfection.In order to detect the efficiency of lentivirus infection,we used qPCR and thin layer liquid chromatography(TLC)to identify the SMS2 expression level and SMS activity,respectively.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was employed to assay the content of sphingomyelin(SM),ceramide(Cer)in cells.Cell were incubated with lysenin and then detected cell viability.the SM level of plasma membrane is negatively correlated with cell viability.Results:1.The recombinant lentiviral vectors of SMS2 overexpression or knockdown were successfully established and integrated into the genome of INS-1 cells,respectively.Meanwhile,the negative control was set.2.Compared with the control group,the SMS expression level of INS-1 shRNA-SMS2 cells was significantly decreased,and the SMS expression level in INS-1 SGMS2 cells was markedly increased.3.Compared with INS-1 shRNA-NC cells,the SMS activity of INS-1 shRNA-SMS2 cells decreased 80%,and the total SM level decreased significantly and the general ceramide increased obviously,of which the C16:0,C18:0,C24:0 species of Cer were given priority to increase.On the other hand,the SMS activity elevated 4.4 folds and the total SM content cells were also elevated significantly in INS-1 SGMS2 cells(total SM:2306.52±37.20 vs.1637.34±61.62 vs.2511.04±387.11 pmol·lig-1·ml-1).However,the expected decrease of INS-1 SGMS2 cells has not been detected.(total Cer:602.76±29.731 vs.1146.09±1 30.71 vs.889.29±96.02 pmol·μg-1·ml-1).It may be explained by the dynamic resources of ceramide.4.Compared with control group,the SM of plasma membranes in INS-1 shRNA-SMS2 significantly decreased,but the SM of plasma membranes in INS-1 SGMS2 cell obviously elevated.Conclusions:We successfully constructed the stable cell lines of INS-1 shRNA-SMS2 and INS-1 SGMS2,respectively.In consistent with the reduction of SMS2,the SMS activity,total intracellular SM and the SM in plasma membrane decreased significantly,while the Cer levels increased responsively.The overexpression of SMS2 leads to the increase of total intracellular SM contents and the SM of plasma membrane,but the total Cer increased slightly.Chapter II The effect of sphingoimyelin synthase 2 on the physiological function in INS-1 cellObjective:To explore the impact of SMS2 on physiological function of INS-1 cell.Methods:We used cell counting kit-8(CCK-8)and flow cytometry to detect the effects of SMS2 deficiency on the cell viability,differentiation and survival.We determined the effect of SMS2 on cell migration ability through transwell experiment.We utilized transmission electron microscope(TEM)to observe the cellular structure and assayed intracellular methane dicarboxylic aldehyde(MDA)content and ATP production.We measured K+-stimulated insulin secretion(KSIS)and cell immune fluorescent to probe the impact of SMS2 on insulin synthesis and insulin secretion in INS-1 cellResults:1.Incubated with lysenin,comared with the control group,we found that the SM content of plasma membrane in INS-1 shRNA-SMS2 cell was lower than the control group and the SM content of plasma membrane in INS-1 shRNA-SMS2 cell was higher.2.Compared with the control group,knockdown of SMS2 expression in INS-1 cell lead to decrease the growth ability and increase the rate of cell apoptosis.However,the differentiation ability of INS-1 cell was not influenced by SMS2 downregulation.The growth ability of INS-1 SGMS2 cell was upregulated.3.Compared with INS-1 shRNA-NC cell,the cell migration potential in INS-1 shRNA-SMS2 cell was reduced significantly,the INS-1 SGMS2 cell was not influenced.4.Compared with control group,the insulin synthesis and KSIS were not influenced by the SMS2 knockdowm and overexpression.However,the mRNA expression level and protein content of glucose transporter 2(GLUT2)in INS-1 shRNA-SMS2 cell was lower than the control group and the INS-1 SGMS2 cell.6.We observed merely no difference in cellular integrity and the morphology and distribution of the organelles among theses groups.5.The ATP synthesis ability and the content of oxidative stress products in INS-1 shRNA-SMS2 group and INS-1 SGMS2 group were consistent with INS-1 shRNA-NC group.Conclusions:1.The change of SMS2 expression had no effect on insulin synthesis and the K+-stimulated insulin secretion,but resulted in the decrease of GLUT2,which could be impaired glucose-stimulated insulin secrerion and need to conduct several experiments in cells,which has the ability of glucose-stimulated insulin secrerion.2.The lack of SMS2 expression in INS-1 cells decreased the viability of cell growth,inhibited the potential of cell migration and increase the apoptosis rate.3.The ceramide increment of C16:0,C18:0,C24:0 species didn’t cause toxic effects in rat islet beta cell.